Targeting protein-protein interactions (PPI's) has proven to be a difficult task for conventional medicinal chemistry. While small molecules (metabolically stable and often cell-penetrant) and large biologics (high target affinity and specificity) each offer distinct advantages, peptides are advantageous in that they may represent the intersection of these promising characteristics. However, developing short peptides into useful probes and therapeutic leads remains a difficult challenge for medicinal chemists. Structural rigidification is a powerful strategy for improving the properties of short peptides, which suffer from poor target affinity and selectivity due to their conformational flexibility. Conformational restriction of linear and cyclic peptides has been shown to be an effective tool in the development of potent and selective biological probes. Intramolecular cross-linking represents one such class of modifications that has led to improvements in affinity, selectivity and resistance to proteolytic degradation. Examples of this cross-linking strategy are abundant in nature, and have notably been applied to the stapling of alpha helices. However, there are very few examples in the literature of applying intramolecular cross-linking to the structural preorganization of cyclic peptides. We propose that this strategy can improve the pharmacological profile of an already promising class of molecular recognition agents. We have applied the strategy of intramolecular cross-linking to low-molecular weight (11-residue) head-to-tail cyclic peptides that target the Src homology 2 (SH2) domain of Grb2, a validated cancer target. Preliminary investigations have been conducted by the PI that demonstrate the application of this strategy to the development of a bicyclic peptide inhibitor of the Grb2-SH2 domain that features enhanced affinity, selectivity and resistance to proteolytic degradation. In this proposal, we seek to expand on this approach by developing improved inhibitors of Grb2-SH2 using structure-based design. We will incorporate detailed information obtained from solving X-ray crystal structures of Grb2 in complex with our bicyclic inhibitors in order to inform inhibitor design. We also seek to solve the NMR solution structures of these bicyclic peptide inhibitors, so as to correlate promising pharmacological features with specific structural characteristics. We will probe the cell-penetrating capabilities of our inhibitors, as well as the cellular phenotype these compounds exhibit in live cancer cells. Finally, to demonstrate the applicability of our platform, we seek to utilize this peptide bicycle technology for the development of inhibitors of protein tyrosine phosphatase 1B (PTP1B), a validated target of obesity and type II diabetes. We propose herein that conformationally-constrained bicyclic peptides offer a promising new therapeutic modality to combating difficult-to-target protein-protein interactions.